The Diaphanous-related Formins (DRFs) are important effector molecules of Rho GTPases, and control multiple aspects of the form and function of the cytoskeleton, exerting particularly tight control over the actin cytoskeleton. This situates the DRFs in a key position to influence neuronal morphological differentiation. Essentially nothing is known, however, about expression, localization, or function of these signaling proteins in neuronal cells. The overall goal of these studies is to identify and define the roles of the DRFs in neuronal morphogenesis. Specific antibodies will be used on protein blots to quantify DRF protein expression in the developing brain and in cell culture models of neuronal differentiation. Immunofluorescence techniques will be employed to localize DRF proteins in cellular compartments during neuronal process outgrowth, focusing on areas of cytoskeletal dynamics such as the growth cone. Exogenous expression of mutant DRF proteins will be used to manipulate DRF activity and thereby to test the hypothesis that DRF function positively influences neurite outgrowth. Co-immunoprecipitation experiments will examine the association of DRFs with a major binding partner, the elongation factor eEFla, which is an abundant actin-binding protein, in neuronal cells before and during process outgrowth, and will examine the role of the Rho-regulated Rho kinase in the formation of this interaction. The results will be used to construct a model of DRF function in neurons and will shed light on the role of one major DRF binding partner, eEFla. The findings will help uncover the cellular mechanisms that underlie neuronal process outgrowth, a developmental function that is recapitulated in neural regeneration. Understanding these mechanisms is critical in the search for therapeutic tools to use in cases of degenerative disease or trauma in nervous system.